Nigerian-TCGA Differential Expression Analysis Using Limma/Voom Long Non-Coding Genes Only
Padma Sheila Rajagopal, MD MPH
6/7/2020
Last updated: 2020-06-08
Checks: 5 2
Knit directory: ~/Research-Local/2019-rnaseq/TCGA-Nigerian-RNAseq/
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- Preparing the sampleTable using HTSeq raw counts
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#Translation from HTSeq raw counts -> Count Matrix I have 84 TCGA patients with whole-genome sequencing data and RNAseq data as well as 95 Nigerian patients with RNA-seq data. Raw counts were initially processed using HTSeq, so HTSeq data is being formatted for use with DESeq2 and limma-voom.
sampleConditionPAM50
sampleConditionrace Basal Her2 LumA LumB Normal PAM_other
Nigerian 29 24 14 16 6 6
TCGA_black 23 0 4 4 0 0
TCGA_other 0 0 0 0 0 14
TCGA_white 17 5 8 9 0 0
Warning: The above code chunk cached its results, but it won’t be re-run if previous chunks it depends on are updated. If you need to use caching, it is highly recommended to also set knitr::opts_chunk$set(autodep = TRUE) at the top of the file (in a chunk that is not cached). Alternatively, you can customize the option dependson for each individual chunk that is cached. Using either autodep or dependson will remove this warning. See the knitr cache options for more details.
#Quantile normalization Please refer to: https://parajago.github.io/TCGA-Nigerian-RNAseq/NigerianTCGArawcountsDeSeq2-pc2.html regarding comparison between the Nigerian and TCGA data sets and why quantile normalization under the limma-voom approach was chosen for primary differential expression analysis.
##Data visualization
Warning: The above code chunk cached its results, but it won’t be re-run if previous chunks it depends on are updated. If you need to use caching, it is highly recommended to also set knitr::opts_chunk$set(autodep = TRUE) at the top of the file (in a chunk that is not cached). Alternatively, you can customize the option dependson for each individual chunk that is cached. Using either autodep or dependson will remove this warning. See the knitr cache options for more details.
#Differential expression setup
Warning: The above code chunk cached its results, but it won’t be re-run if previous chunks it depends on are updated. If you need to use caching, it is highly recommended to also set knitr::opts_chunk$set(autodep = TRUE) at the top of the file (in a chunk that is not cached). Alternatively, you can customize the option dependson for each individual chunk that is cached. Using either autodep or dependson will remove this warning. See the knitr cache options for more details.
##DE: Nigerian/TCGA White - Basal
[1] 5172 46
TCGA_white.Basal - Nigerian.Basal
Down 492
NotSig 4562
Up 118
Warning: The above code chunk cached its results, but it won’t be re-run if previous chunks it depends on are updated. If you need to use caching, it is highly recommended to also set knitr::opts_chunk$set(autodep = TRUE) at the top of the file (in a chunk that is not cached). Alternatively, you can customize the option dependson for each individual chunk that is cached. Using either autodep or dependson will remove this warning. See the knitr cache options for more details.
##DE: Nigerian/TCGA Black - Basal
[1] 5305 52
TCGA_black.Basal - Nigerian.Basal
Down 398
NotSig 4786
Up 121
Warning: The above code chunk cached its results, but it won’t be re-run if previous chunks it depends on are updated. If you need to use caching, it is highly recommended to also set knitr::opts_chunk$set(autodep = TRUE) at the top of the file (in a chunk that is not cached). Alternatively, you can customize the option dependson for each individual chunk that is cached. Using either autodep or dependson will remove this warning. See the knitr cache options for more details.
##DE: Nigerian/TCGA White - HER2 (no TCGA Black HER2+ patients)
[1] 4652 29
TCGA_white.Her2 - Nigerian.Her2
Down 620
NotSig 3897
Up 135
Warning: The above code chunk cached its results, but it won’t be re-run if previous chunks it depends on are updated. If you need to use caching, it is highly recommended to also set knitr::opts_chunk$set(autodep = TRUE) at the top of the file (in a chunk that is not cached). Alternatively, you can customize the option dependson for each individual chunk that is cached. Using either autodep or dependson will remove this warning. See the knitr cache options for more details.
##DE: Nigerian/TCGA White - LumA
[1] 4211 22
TCGA_white.LumA - Nigerian.LumA
Down 367
NotSig 3780
Up 64
Warning: The above code chunk cached its results, but it won’t be re-run if previous chunks it depends on are updated. If you need to use caching, it is highly recommended to also set knitr::opts_chunk$set(autodep = TRUE) at the top of the file (in a chunk that is not cached). Alternatively, you can customize the option dependson for each individual chunk that is cached. Using either autodep or dependson will remove this warning. See the knitr cache options for more details.
##DE: Nigerian/TCGA Black - LumA
[1] 4056 18
TCGA_black.LumA - Nigerian.LumA
Down 370
NotSig 3634
Up 52
Warning: The above code chunk cached its results, but it won’t be re-run if previous chunks it depends on are updated. If you need to use caching, it is highly recommended to also set knitr::opts_chunk$set(autodep = TRUE) at the top of the file (in a chunk that is not cached). Alternatively, you can customize the option dependson for each individual chunk that is cached. Using either autodep or dependson will remove this warning. See the knitr cache options for more details.
##DE: Nigerian/TCGA White - LumB
[1] 4272 25
TCGA_white.LumB - Nigerian.LumB
Down 285
NotSig 3933
Up 54
Warning: The above code chunk cached its results, but it won’t be re-run if previous chunks it depends on are updated. If you need to use caching, it is highly recommended to also set knitr::opts_chunk$set(autodep = TRUE) at the top of the file (in a chunk that is not cached). Alternatively, you can customize the option dependson for each individual chunk that is cached. Using either autodep or dependson will remove this warning. See the knitr cache options for more details.
##DE: Nigerian/TCGA Black - LumB
[1] 4081 20
TCGA_black.LumB - Nigerian.LumB
Down 333
NotSig 3630
Up 118
Warning: The above code chunk cached its results, but it won’t be re-run if previous chunks it depends on are updated. If you need to use caching, it is highly recommended to also set knitr::opts_chunk$set(autodep = TRUE) at the top of the file (in a chunk that is not cached). Alternatively, you can customize the option dependson for each individual chunk that is cached. Using either autodep or dependson will remove this warning. See the knitr cache options for more details.
sessionInfo()R version 3.6.3 (2020-02-29)
Platform: x86_64-apple-darwin15.6.0 (64-bit)
Running under: macOS Sierra 10.12.6
Matrix products: default
BLAS: /Library/Frameworks/R.framework/Versions/3.6/Resources/lib/libRblas.0.dylib
LAPACK: /Library/Frameworks/R.framework/Versions/3.6/Resources/lib/libRlapack.dylib
locale:
[1] en_US.UTF-8/en_US.UTF-8/en_US.UTF-8/C/en_US.UTF-8/en_US.UTF-8
attached base packages:
[1] parallel stats4 grid stats graphics grDevices utils
[8] datasets methods base
other attached packages:
[1] msigdbr_7.1.1 fgsea_1.10.0
[3] Rcpp_1.0.1 AnnotationHub_2.16.1
[5] BiocFileCache_1.8.0 dbplyr_1.4.2
[7] Glimma_1.12.0 RColorBrewer_1.1-2
[9] preprocessCore_1.46.0 ashr_2.2-32
[11] ggfortify_0.4.7 calibrate_1.7.2
[13] MASS_7.3-51.5 sva_3.32.1
[15] mgcv_1.8-31 nlme_3.1-144
[17] EnsDb.Hsapiens.v75_2.99.0 ensembldb_2.8.0
[19] AnnotationFilter_1.8.0 GenomicFeatures_1.36.4
[21] hexbin_1.27.3 stringi_1.4.3
[23] dplyr_0.8.3 affy_1.62.0
[25] checkmate_1.9.3 pathview_1.24.0
[27] org.Hs.eg.db_3.8.2 AnnotationDbi_1.46.0
[29] clusterProfiler_3.12.0 pheatmap_1.0.12
[31] genefilter_1.66.0 vsn_3.52.0
[33] RUVSeq_1.18.0 EDASeq_2.18.0
[35] ShortRead_1.42.0 GenomicAlignments_1.20.0
[37] Rsamtools_2.0.0 Biostrings_2.52.0
[39] XVector_0.24.0 DESeq2_1.24.0
[41] SummarizedExperiment_1.14.0 DelayedArray_0.10.0
[43] BiocParallel_1.18.0 matrixStats_0.54.0
[45] Biobase_2.44.0 GenomicRanges_1.36.0
[47] GenomeInfoDb_1.20.0 IRanges_2.18.1
[49] S4Vectors_0.22.0 BiocGenerics_0.30.0
[51] edgeR_3.26.4 limma_3.40.2
[53] ggbiplot_0.55 scales_1.0.0
[55] plyr_1.8.5 ggplot2_3.2.1
[57] gplots_3.0.3
loaded via a namespace (and not attached):
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[117] Hmisc_4.2-0 pillar_1.4.2
[119] htmltools_0.3.6 mime_0.7
[121] glue_1.4.0 DESeq_1.36.0
[123] interactiveDisplayBase_1.22.0 codetools_0.2-16
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[129] GO.db_3.8.2 survival_3.1-8
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